Antibiotic Sensitivity Testing
On this page
Direct answer
A zone diameter means nothing on its own — it acquires meaning only from the standardisation that produced it. The Kirby-Bauer disc diffusion method fixes every variable: Mueller-Hinton agar poured 4 mm deep at pH 7.2-7.4, an inoculum matched to the 0.5 McFarland standard (about 1.5 × 10⁸ colony-forming units per mL), discs applied within 15 minutes of inoculation, incubation at 35 ± 2 degrees C for 16-18 hours, and zone measurements interpreted against current CLSI breakpoints as susceptible, intermediate or resistant. The zone is only a proxy; the true measurand is the minimum inhibitory concentration (MIC), determined directly by broth microdilution (the reference method), agar dilution or gradient diffusion. Break the standardisation — thin agar, heavy inoculum, stale discs, wrong temperature — and the category on the report becomes fiction.
What you must remember
- 0.5 McFarland barium sulphate standard equals roughly 1.5 × 10⁸ CFU/mL; match the inoculum turbidity visually or photometrically and use the suspension within about 15 minutes.
- Mueller-Hinton agar: 4 mm depth — thinner plates give falsely large zones, thicker plates falsely small; pH 7.2-7.4; add 5 per cent sheep blood for streptococci, use HTM for Haemophilus and supplemented GC agar for gonococci.
- Discs go on within 15 minutes of lawn inoculation (distribute so zones do not overlap), plates incubate inverted at 35 ± 2 degrees C for 16-18 hours, with 24 hours for vancomycin and oxacillin screens.
- Categories per CLSI zone charts (updated annually): susceptible, intermediate — "susceptible, increased exposure", meaning achievable with higher dosing — and resistant; never extrapolate between drugs.
- Resistance confirmation is phenotypic: MRSA by cefoxitin disc or oxacillin screening; ESBL by a ceftazidime-clavulanate zone at least 5 mm larger than ceftazidime alone; inducible clindamycin resistance by the D test.
- MIC methods: broth microdilution (the reference method), agar dilution and E-test gradient strips — needed when precise quantitation guides therapy, as in endocarditis or resistant Gram-negative infections.
- Quality control strains per CLSI: Staphylococcus aureus ATCC 25923 (discs), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, run with each new lot and weekly.
- Definitions escalating under Indian stewardship programmes (ICMR AMR surveillance): multidrug-resistant (non-susceptible to at least one agent in three or more classes), extensively drug-resistant, and pandrug-resistant.
A D test that changed the report
A Staphylococcus aureus from a diabetic foot ulcer reports erythromycin resistant (zone 6 mm) with clindamycin susceptible (zone 24 mm) — on paper, clindamycin is an oral option. The alert technologist knows erythromycin resistance can hide inducible erm-mediated clindamycin resistance that routine testing misses, so the D test is set: erythromycin and clindamycin discs placed at the standard spacing, typically 15-26 mm apart. After incubation, the clindamycin zone shows a flattened, D-shaped edge facing the erythromycin disc — induction proven. The report changes to clindamycin resistant, and a therapy that would have failed is replaced.
The negative D test matters equally: a uniform circular clindamycin zone with an erythromycin-resistant isolate usually signals the efflux mechanism (msrA), which does not induce — clindamycin remains usable. One extra disc, one night's incubation — that is stewardship in action, and the framing examiners reward when asked why the D test exists. The same logic of confirmation extends to ESBLs: a cephalosporin zone shrunk on its own proves nothing until the clavulanate-enhanced comparison demonstrates enzyme inhibition.
Where students slip
Reading zones after 24 hours "to be sure" voids the breakpoints the chart was built on — the category applies only to the method as specified. Interpreting intermediate as resistant wastes a usable drug: "susceptible, increased exposure" invites dose optimisation, and examiners probe this vocabulary. Practical marks die quietly on inoculum errors: a suspension too heavy shrinks every zone toward resistance, and a swarming Proteus plate or overlapping discs makes measurement arbitrary — correct with a pure culture, correct turbidity and sensible disc placement.
Frequently asked questions
What is the 0.5 McFarland standard and why does it matter?
A barium sulphate turbidity equivalent to about 1.5 × 10⁸ CFU/mL that standardises inoculum density — the single variable most responsible for zone size accuracy.
State the medium, depth and incubation conditions for Kirby-Bauer testing.
Mueller-Hinton agar 4 mm deep at pH 7.2-7.4, discs within 15 minutes of inoculation, incubated inverted at 35 ± 2 degrees C for 16-18 hours (24 for vancomycin and oxacillin).
What is the D test and when is clindamycin reported resistant?
Erythromycin and clindamycin discs placed adjacently; a flattened D-shaped clindamycin zone indicates inducible erm-mediated resistance, and clindamycin is reported resistant despite a normal zone.
How is ESBL production confirmed phenotypically?
The ceftazidime-clavulanate combination zone exceeding the ceftazidime-alone zone by 5 mm or more demonstrates extended-spectrum beta-lactamase activity.
Which methods determine MIC directly?
Broth microdilution (the reference), agar dilution and gradient diffusion (E-test), reported with a category interpretation in mg/L.
Which QC strains accompany AST, and how often?
Standard CLSI strains — S. aureus ATCC 25923, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 — tested weekly and with every new disc lot or medium batch.